Parkinson's disease (PD) and several other neurodegenerative diseases are degenerative disorders of the central nervous system and are both chronic and progressive. The prevalence of PD in Europeans is 1.6% in persons over 65 years of age. However, more than 10% of the patients are diagnosed before the age of 50. In 1990, an estimated 4 million people were suffering from PD. It is characterized by muscle rigidity, tremor, a slowing of physical movement (bradykinesia) and, in extreme cases, a loss of physical movement (akinesia). The primary symptoms are the results of decreased stimulation of the motor cortex by the basal ganglia, normally caused by the insufficient formation and action of dopamine, which is produced in the dopaminergic neurons of the brain. Secondary symptoms may include high level cognitive dysfunction and subtle language problems. Typical other symptoms include disorders of mood, behavior, thinking, and sensation (non-motor symptoms). Patients' individual symptoms may be quite dissimilar and progression of the disease is also distinctly individual.
The symptoms of Parkinson's disease result from the loss of dopaminergic cells in the region of the substantia nigra pars compacta. These neurons project to the striatum and their loss leads to alterations in the activity of the neural circuits within the basal ganglia that regulate movement, in essence an inhibition of the direct pathway (facilitating movement) and excitation of the indirect pathway (inhibiting movement). The lack of dopamine results in increased inhibition of the ventral anterior nucleus of the thalamus, which sends excitatory projections to the motor cortex, thus leading to hypokinesia.
The pathological hallmark feature of Parkinson's disease (PD) and several other neurodegenerative disorders is the deposition of intracytoplasmic neuronal inclusions termed Lewy bodies. The major component of Lewy bodies are amyloid fibrils of the protein alpha-synuclein (alpha-S). Protecting neurons from the toxicity of alpha-synuclein is a promising strategy for treating these diseases.
Related diseases (sometimes called Parkinson-plus diseases) include dementia with Lewy bodies (DLB). While idiopathic Parkinson's disease patients also have Lewy bodies in their brain tissue, the distribution is denser and more widespread in DLB. Even so, the relationship between Parkinson disease, Parkinson disease with dementia (PDD), and dementia with Lewy bodies (DLB) might be most accurately conceptualized as a spectrum, with a discrete area of overlap between each of the three disorders. The common involvement of alpha-synuclein in diseases such as PD, PDD, multiple system atrophy and the Lewy body variant of Alzheimer's disease has led to a classification of these disease under the term synucleinopathies.
Mutations of alpha-S associated with familial PD (A30P, A53T, E46K) have an increased aggregation propensity in vitro (US 2007/0213253), in agreement with aggregation of alpha-S into fibrillar Lewy bodies in vivo. However, the role different aggregated alpha-S species play for neurotoxicity in vivo is unclear. Loss of dopaminergic terminals was observed in the presence of non-fibrillar alpha-S inclusions in one line of A53T alpha-S transgenic mice (Masliah et al. Science 287, 1265-1269 (2000)), raising the possibility that pre-fibrillar intermediates in the alpha-S aggregation process may be pathogenic (Lashuel & Lansbury Q Rev Biophys 39, 167-201 (2006)), potentially by pore formation in cell membranes. However, transgenic mice overexpressing A30P alpha-S failed to exhibit neurodegeneration (Lee et al. Proc Natl Acad Sci USA 99, 8968-8973 (2002)), despite the fact that A30P alpha-S delays conversion of pre-fibrillar aggregates to fibrils (Conway et al., supra). Thus, the A30P alpha-S mice provided in vivo support that pre-fibrillar alpha-S is not the primary toxic moiety.
US 2007/0192879 and WO 2008/063779 describe animal models and cell models overexpressing alpha-S. The only disclosed mutations are A30P, A53T and E46K, which are known to show an increased aggregation propensity.
US 2007/0213253 disclose several synuclein mutants having aggregation-inhibitory activity. Those mutants are supposed to be capable of inhibiting aggregation of wt alpha-S.
Also, Koo et al. Biochem. Biophys. Res. Comm. 368, 772-778 (2008) describe substitutions of alpha-synuclein which exhibit the capability to influence fibril formation of the protein.
Zhou et al. J. Biol. Chem. 283(15), 9863-9870 (2008) teaches tyrosine to cysteine substitutions in human alpha-synuclein showing enhanced alpha-synuclein fibril formation and neurotoxicity.
Thus, there is a need in the art for alpha-synuclein mutants that can be used in in vivo and in vitro screening assays in order to identify substances that can prevent or reduce the toxicity of alpha-synuclein, which plays a pivotal role in the pathology of synucleinopathies.